Extracellular matrix (ECM) assembly is an essential part of development, tissue homeostasis, aging, wound healing, and initiation and progression of disease. The physical and mechanical properties of the ECM are emerging as critical regulators of these processes through their effects on cell organization and signaling. During development and in pathologies such as cancer, fibrosis, and connective tissue abnormalities, the composition and the physical state of the ECM can vary dramatically. To develop treatments to prevent or reverse the effects of abnormal ECM, we need to understand the molecular interactions that assemble a tissue-appropriate matrix and how these are affected in disease. The overall goal of this proposal is to determine how the assembly of the pericellular matrix supports and directs the subsequent assembly of additional ECM components to build a tissue-appropriate definitive matrix. Fibronectin (FN) is a ubiquitous ECM protein that makes an essential connection between cell surface receptors and other ECM components. FN is assembled into a fibrillar ECM via a cell-mediated process that involves FN conformational changes to promote FN-FN interactions and culminates with the definitive FN matrix that can be identified by insolubility in the detergent deoxycholate (DOC). A critical question for understanding tissue-appropriate ECM assembly is the effect of compliance since matrix assembly occurs in tissues that differ significantly in stiffness. In Aim 1 we will test the hypothesis that stiffness of the pericellular ECM governs FN matrix assembly by regulating one or more key steps in the assembly process. We will analyze the major steps of assembly (integrin binding, FN conformational changes, and DOC insolubility) using polyacrylamide gel substrates of defined elastic modulus and will test the idea that FN conformation differs in matrices assembled under different stiffness conditions using a conformation-sensitive antibody. The insoluble FN matrix forms the foundation for assembly of other ECM proteins, including some collagens, yet we have limited understanding of its structure. In Aim 2, we will test the hypothesis that specific FN-FN interactions mediate the final step in assembly of the definitive DOC- insoluble matrix. Mutant recombinant FNs will be used to identify important interacting residues and a novel mass spectrometry screen will be applied to identify other FN domains involved in DOC-insoluble interactions. To understand the interplay between FN assembly, tissue stiffness, and incorporation of other ECM proteins, in Aim 3 we will test the hypothesis that FN matrix controls both cell fate and deposition of collagens in a tissue differentiation model, in vitro chondrogenesis. We will manipulate the level of insoluble FN matrix and its stiffness and determine the effects on differentiation markers and assembly of collagens during chondrogenic differentiation of mesenchymal stem cells. The information obtained through the proposed studies will provide novel insights into the mechanisms of normal matrix assembly and will suggest ways in which disease can change assembly to cause abnormal ECM accumulation as in fibrosis, scar formation, and cancer.